Structural element, a mediator of adhesion, and an extracellular signaling molecule. The quantity and the qualities of the HA polysaccharide modulate cell behavior during development, intercellular adhesion and recognition, locomotion, and neovascularization. The long-term goal of this investigation is to elucidate the structure and the mechanism of action of the HA synthase enzymes responsible for production of HA in humans. The biosynthesis of HA in vertebrates has been studied for decades with little success until 1996, when a family of eukaryotic HA synthases was identified. These enzymes are novel and interesting glycosyltransferases because they transfer two different sugar groups during polymerization; almost all other enzymes transfer only one sugar. The molecular and biochemical details of HA biosynthesis in vertebrates are lacking. Therefore, the major goal of this five-year project period is to characterize several HA synthases from Xenopus and humans. The investigators have produced functional recombinant xenopus HA synthase enzyme in a background-free system. The specific aims of this project are; 1) To analyze the kinetics of the eukaryotic HA synthases. Classical biochemical studies will reveal the manner in which the enzyme polymerizes its UDP-sugar precursors. 2) To characterize structurally the eukaryotic HA synthases. Radiation inactivation and mass spectrometry will be used to define the functional unit and monomer sizes, respectively. 3) To identify the precursor binding sites of the HA synthases. photoaffinity and chemical labeling studies will be utilized ot tag regions of the enzyme that interact with the UDP-sugar substrates. 4) To determine the functional residues of the HA synthase. The residues that are conserved among HA synthases will be altered by site-directed mutagenesis; in particular, certain cysteine residues will be mutated due to their potential roles in catalysis and in disulfide bond formation. The essential residues identified in aim 3 will also be confirmed by mutagenesis studies. The enzymological and structural information may allow the generation of a working model describing the catalytic mechanism of these enzymes.